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Bulletin UASVM, Veterinary Medicine 68(2)/2011
pISSN 1843-5270; eISSN 1843-5378
Protective Effects of Some Bioactive Compounds Treatment upon Some Histochemical
and Biochemical Parametters in the Brain Rabbitt Fetuses Prenatally Exposed to Some
Excitotoxins
Mihai CRISTESCU1), Ioana ROMAN2)
1)
University of Agricultural Sciences and Veterinary Medicine, Cluj-Napoca,
e-mail: [email protected]
2)
National Institute of Research and Development for Biological Sciences, branch
Institute of Biological Research, Cluj-Napoca
Abstract. Administration of L-monosodium glutamate (MSG), L-aspartate (ASP) and
L-cysteine (CYS), during 6-18 days of gestation (organogenetic period of gestation) in rabbits
induced a reduction of the nuclear DNA and RNA fluorescence in all neuronal brain
populations, and a significant decrease of DNA and protein quantity in total brain
homogenate, comparative to control group. Administration of MSG, ASP and CYS associed
with a vitamin-mineral bioactive complex during 6-18 days of gestation shows a moderate
diminution of the DNA and RNA fluorescence, in all neuronal brain structures, and a
moderate decrease of DNA and protein quantity comparative to control group.
Keywords: excitotoxins, bioactive, gestation, fetuses, DNA, protein, quantity.
INTRODUCTION
Excitotoxins are biochemical substances (usually amino acids, amino acid analogs, or
amino acid derivatives) that can react with specialized neuronal receptors - glutamate
receptors - in the brain or spinal cord that can be responsible for causing death or injury to a
wide array of neurons.
There are a large and growing number of clinicians and basic scientists who believe
that excitotoxins play a critical role in the development of several neurological disorders
including migraines, seizures, abnormal neural development, neuropsychiatric disorders,
learning disorders in children, AIDS dementia, episodic violence, lyme borreliosis, hepatic
encephalopathy, specific types of obesity, neurodegenerative diseases, such as ALS,
Parkinson's disease, Alzheimer's disease, Huntington's disease, and olivopontocerebellar
degeneration, (Blaylock, 1997; Garattini, 2000; Lombardi, 2002; Olney, 1994).
These amino acids are known to trigger the formation of enormous storms of free
radicals, leading to prolonged lipid peroxidation (oxidation of cell membranes and
membranes within the cell), associated with DNA injury. Both of these effects, causes free
radical generation, DNA damage (DNA fragmentation), and DNA mutation in neurons of the
hippocampus, hypothalamus and cerebral cortex, (Gilliams-Francis et al., 2003; Kure et al.,
1991; Puica, Craciun and Rusu, 2004; Puica et al., 2007ab, 2008; White and Reynolds, 1996).
Glutamate is one of the more commonly known excitotoxins, but over seventy have
thus far been identified. A recent study found that newborns exposed to monosodium
glutamate (the sodium salt of glutamate - MSG) from day 1-10 still had a free radical
elevation of 56% eighty days later, (Wang et al., 2008).
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Glutamate neurons make up an extensive network throughout the cortex,
hippocampus, striatum, thalamus, hypothalamus, cerebellum, and visual/auditory system. As
a consequence, glutamate neurotransmission is essential for cognition, memory, movement,
and sensation (especially taste, sight, and hearing). Glutamate and its biochemical “cousin”
aspartic acid or aspartate, are the two most plentiful amino acids in the brain. Aspartate is also
a major excitatory transmitter and aspartate can activate neurons in place of glutamate.
Glutamate, as a physiologic excitatory neurotransmitter in the brain exists in the extracellular
fluid only in very, very small concentrations. When the concentration of glutamate rises above
normal levels, the neurons begin to fire abnormally. At higher concentrations, the cells
undergo a process of delayed cell death, called “excitotoxicity”. Glutamate binds to receptors
on neurons and over-activates them, causing the brain cells to die. That is, they are excited to
death, (Blaylock, 1997; Olney, 1994).
Release of glutamate, the major excitatory amino acid neurotransmitter in the brain, is
one of the primary events in the excitotoxic cascade. Release of glutamate combined with
reversal of glutamate uptake pumps in nerve terminals causes glutamate levels to increase
from 10-1000x baseline levels in the brain's extracellular space during ischemia, traumatic
brain injury, and hypoglycemia in animal models and in humans. High levels of extracellular
glutamate trigger opening of N-methyl-D-asparate (NMDA) and non-NMDA glutamate
channels, allowing calcium and sodium to flood into neurons. Energy failure in mitochondria
facilitates opening of NMDA channels by lowering the membrane potential and reducing their
blockade by magnesium that resides within the channel at normal membrane potentials.
Calcium entry into the cytoplasm triggers several events including activation of lipases,
proteases, and nucleases that can destroy the neuron's cellular machinery. Excessive Ca2+ ions
also activates various proteases (protein-digesting enzymes) which can digest various cell
proteins, including tubulin, microtubule-proteins, spectrin, and others. Ca2+ can also activate
nuclear enzymes (endonucleases) that result in chromatin condensation, DNA fragmentation
and nuclear breakdown, i.e. apoptosis, or "cell suicide" (Gilliams-Francis et al., 2003; Lipton
and Rosenberg, 1994; Wang et al., 2008).
Generation of reactive oxygen free radicals including the messenger molecule nitric
oxide can also have additional destructive effects. Thus, glutamate mediated intracellular
calcium accumulation and free radical generation are thought to be major mechanisms that
contribute to cell death in brain injury, (Blaylock, 2002; Finiels et al., 2002).
Drugs that block glutamate receptors and the excitotoxic cascade of downstream
events can protect the brain from injury and show promise for clinical application in infants
and children, (Tastekin et al., 2005).
It is now known that glutamate (and therefore aspartate) is the major neurotransmitter
in the hypothalamus and therefore excess concentrations may affect the hippocampic
structures and all of the various nuclei in the hypothalamus. This means that virtually every
function of the hypothalamus is vulnerable to excitotoxin damage, both subtle and acutely
dramatic, depending on the dose. In normal everyday life we are exposed to numerous
excitotoxins added to foods and drinks, in the form of MSG, cysteine and Aspartame. Several
studies have shown that these toxic doses are synergistic, that is, they are more than just the
sum effect of each excitotoxin. Therefore, a meal of MSG laden soup, a diet cola and foods
with hydrolyzed vegetable protein and natural flavoring, could easily damage the
hypothalamus, as well as other portions of the nervous system (thalamic nuclei and
hippocampus). During pregnancy, the deleterious effects of the excitotoxins could be even
more devastating, since it will affect the development of the brain itself, (Blaylock, 2002;
Stegink et al., 1982).
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Aspartate, another acidic amino acid, is also considered an excitotoxin. When
ingested, aspartate is converted in the liver into glutamic acid, the toxic component of
monosodium glutamate.
A newer addition to the family of excitotoxin food additives is L-cysteine, which in
the body is converted to the powerful excitotoxin cysteine sulfinic acid. L-cysteine is being
added to certain breads as a dough conditioner. Experimentally, MSG or aspartate can
produce the same brain lesions using hydrolyzed vegetable protein. A growing list of
excitotoxins is being discovered, including several that are found naturally. For example, Lcysteine (CYS) is a very powerful excitotoxin. Recently, it has been added to certain bread
dough and is sold in health food stores as a supplement. Homocysteine, a metabolic
derivative, is also an excitotoxin. Interestingly, elevated blood levels of homocysteine has
recently been shown to be a major, if not the major, indicator of cardiovascular disease and
stroke. Equally interesting, is the finding that elevated levels of CYS have also been
implicated in neurodevelopmental disorders, especially anencephaly and spinal dysraphism
(neural tube defects), (Blaylock, 2002; Puica et al., 2004).
Glutamate/aspartate mediated intracellular calcium accumulation and free radical
generation are thought to be major mechanisms that contribute to cell death in hypoxicischemic brain injury. Medical research is focusing more and more on ways to combat
excitotoxicity. Yet the few available clinical or experimental excitotoxicity-blocking drugs so
far discovered have significant side effect potential - they may block normal, essential GLU
neurotransmission as well as excitotoxicity. Fortunately, a review of the basics of GLU
excitotoxicity reveals a host of preventative nutritional/life extension drug strategies that will
minimize or even eliminate the excitotoxic "dark side" of GLU/ASP and CYS.
For this reason, various glutamate receptor antagonists and antioxidants have been
investigated for their therapeutic potential. The present study demonstrates that a vitaminmineral mixture protects against glutamate, aspartate and cysteine-induced neurotoxicity.
Protective effect of this bioactive compound may result from its antioxidant activity because
free radical generation is a common result in either glutamate, aspartate and cysteine-induced
neurotoxicity. Our vitamin-mineral antioxidant mixture merits further investigation as a
therapeutic option in hypoxic-ischemic brain injury of newborn. Most importantly, there are
no studies of the effect of these excitotoxins on the physiology of the nervous system under
conditions of low brain energy supply. Virtually all studies of this problem, other than
behavioral effects, are centered on microscopic pathologic changes and not functional
alterations of either the neurons themselves or of the entire brain itself.
MATERIALS AND METHODS
Nuliparous female rabbits were maintained under standard laboratory conditions.
Females were assigned to treatment groups based on a computerizad body-weight ballanced
random allocation scheme. Females were housed individualy in temperature-controled rooms
with 12 hr light/dark cycles. Animals were fed aproximately 125-150 rat chow per day and
drinking water was available at libitum. Three groups of 8 pregnant rats were used.. The
animals were treated oraly by intragastrical gavage with L-monosodium glutamate (MSG), Laspartate (ASP) and L-cysteine (CYS), and with MSG, ASP and CYS associed with a
vitamin-mineral bioactive complex in the 1-21 days of gestation. Before the administration of
the experimental substances, the animals were “a jeun”.
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Experimental groups. Groups of 8 pregnant female rats weighing 2000 g. were caged
individually in standardized cages. Three experimental groups of were selected:
C group, (control group), received by oral gavage distilled water at 4 ml per kg body weight.
AC group, received an intragastrical gavage of L-monosodium glutamate (MSG) in a dose of
0,15 mg per g body weight (bw), L-aspartate (ASP) in a dose of 0,07 mg/g bw., and Lcysteine (CYS) in a dose of 0,01 mg/g bw. in the 1-21 days of gestation. The substances were
dissolved in distilled watter.
MB group, treated by intragastrical gavage with L-monosodium glutamate (MSG) in a dose
of 0,15 mg per g body weight (bw), L-aspartate (ASP) in a dose of 0,07 mg/g bw., and Lcysteine (CYS) in a dose of 0,01 mg/g bw., associed with a vitamin-mineral bioactive
complex
We cant't give in detail the bioactive moleculae composition, because this is under the
protection of a certificat of invention brevet evaluation During the experimental period, each
female was daily observed, and body weight was measured at frequent period, for the
adjustement of the treatment doses of the analized substances. In the 21 day of gestation the
rats were killed by decapitation. The brain of 8 fetuses from each experimental group was
removed for histochemical and biochemical investigations.
Histochemical analysis of DNA and RNA fluorescence
The purpose of this method was the relative qualitative evaluation of the nucleic acids
(DNA and RNA) of neuronal nuclei from brain cortex, mediobasal hypothalamus (HMB),
hippocampus and choroid plexus structures using Darzynkyewicz et al.(1975), fluorescence
method. The brain of 8 fetuses of each experimental group was removed and immediately
imersed in liquid nitrogen. The brain was sectioned at Shandon London cryotome at 7
microns, and stained with Acridin orange AZ (AO) fluorochrome (a viable DNA stain) in
phosphate buffer (pH=6). The normal DNA exhibit green-yellow fluorescence; the denatured
DNA and RNA exhibit red fluorescence in ultraviolet light (UV).
Results: the undenatured DNA exhibit green-yellow fluorescence and the denatured
DNA and RNA exhibit red fluorescence in ultraviolet light (UV). The relative number of
viable neuronal cells was obtained by scanning the neuronal populations measured from each
sample. Computerized micro-image analysis was applied in order to quantify the numerical
changes of the viable neurons of CA1, CA2 and CA3-layer pyramidal cell processes during
fetal stage of development.
Biochemical investigations
Quantitative determination of DNA from brain tissue
Was performed using Gross-Bellard metod, (1972). In the 19th day of the gestation
the pregnant animals were sacrificed by cervical dislocation, and the brain of fetuses was
rapidly isolated and freeze in liquid nitrogen. 200 mg of fetal brain tissue was prechilled and
homogenated with Potter. The homogenate was suspended in 1,2 ml. digestion buffer per 100
mg. of tissue, and centrifuged 5 min. at 500 x g and discarded the supernatant. The cells were
trypsinized and resuspended in 10 ml. ice-cold PBS, and centrifuged 5 min at 500x g. The
supernatant was discarded, and repeated the centrifugation. The cells sediment were
resuspended in 1 vol. digestion buffer.
Total protein determination
The total proteins from brain homogenate of fetuses were measured according to
Lowry et al., (1951), with the Folin phenol reagent, using bovine serum albumin as a
standard. The cooper-tartrate reagent reactiones with the proteins in the alkaline medium, and
forms an proteine-cooper complex, which reduces the phosphomolibdic-phosphowolframic
reagent at a blue colour compound.
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RESULTS
Results of the histochemical (fluorescence), making evident of the nucleic acids
Staining of live tissues with AO, examination in UV of nuclei that exhibit green
fluorescence, as well as red fluorescence emitted by cytoplasm and nucleol – evidence of
cellular viability, enabled us to obtain specific images of the HMB and CA1 hippocampus
neurons. After counting the total number of cells with a nucleus that emits a yellow-green
fluorescence in UV, cytograms were done of the total relative number of viable cells of the
above-mentioned structures, for a number of 8 animals /experimental group, the arithmetic
average of the total number of cells of each nervous tissue was determined, while the results
are presented in a graph. Examination of tissues under UV light and the evaluation of the
relative number of cells, respectively of viable cells.
C group – in HMB area the distribution of neuronal populations with nuclei that
exhibit green DNA fluorescence, respectively of the neuronal pericarion (RNA) that emits red
fluorescence under UV light is characteristically uniform. After examination of the sections
the following distribution of nuclei numbers (average ± SE) that exhibit green-yellow
fluorescence under UV, respectively of number of viable cells in the above-mentioned
cerebral structures was established: HMB hypothalamic area (4.275 ± 483), (figure 1).
In AC group, there was recorded a reduction, with statistic significance, of the
relative number of viable cells – identified through the green-yellow fluorescence exhibited
by the nuclear DNA, and through the red fluorescence exhibited by the RNA of the neuronal
pericarion. Fragments of red fluorescence that was moderately higher in the pericelular
structures, corresponds to a denaturized DNA, both in the neuronal populations of the
periventricular areas of the HMB hypothalamus, (figure 2).There were determined the
following values of viable cells in the HMB area (2.859 ± 429, p < 0,001).
MB group had a moderate, non significant reduction of the number of cells whose
nucleus (DNA) exhibits green fluorescence, respectively whose neuronal pericarion and
cytoplasm of glial cells that contain RNA emits red fluorescence under UV, in the
periventricular areas of the HMB hypothalamus, (figure 3). The following values of relative
number of viable cells in the cerebral structures, were determined: HMB (4.125,3 ± 457, p >
0,3), (table 1),
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Figure 2 – Significant reduction of the
relative number of viable cells in HMB area
of hypothalamus in AC group,
(magnification 40 x).
Figure 1 – Normal distribution of neuronal
populations in HMB area in C group,
(magnification 40 x)
Figure 3 – Close to normal distribution of HMB viable neurons in MB
group, (magnification 40 x).
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Tab. 1
Average of relative number of viable neurons in HMB area of hypothalamus
Group
n
X ± SE
DNA purity
D%
p
C
8
4.275 ± 483
1,65
-
AC
8
2.859 ± 429
1,53
- 35,6
< 0,001
MB
8
4.125,3 ± 457
1,52
- 15,6
< 0,3
Results of biochemical assesment
Quantitative determination of deoxyribonucleic acid (DNA)
The relative numeric alterations of the viable cells in the examined cerebral structures, are
associated with a quantitative decrease of DNA in the total fetal brain homogenate. Thus,
treatment with MSG, ASP and CYS during embryonic onthogenesis caused a significant
decrease of DNA quantity in AC group (169,85 ± 0,8; - 35,6%; p < 0,001) comparative with
the average value of DNA quantity of the C group (228,18 ± 0,9 mg/100 mg. tissue).
After treatment with MSG, ASP and CYS associated with a vitamin-mineral bioactive
complex (MB group) during the same period of gestation, the average values of DNA
quantity was close to C group: 197,23 ± 0,9 mg/100 mg. tissue, (- 15,6%, p < 0,05),
comparative to C group. The results of the quantitative determination of DNA in the fetal
brain homogentate, the percentage differences between groups and the statistical significance
of the results of all the experimental groups, are presented in table 2.
Table 2
Average values of DNA quantity (µg./100 mg tissue) in fetal brain homogenate
Group
n
X ± SE
DNA purity
D%
p
C
8
228,18 ± 0,9
1,65
-
AC
8
169,85 ± 0,8
1,53
- 35,6
< 0,001
MB
8
197,23 ± 0,9
1,52
- 15,6
< 0,5
Quantitative determination of total brain proteins
Determination of total proteins from total omogenate of fetal brain homogenate, has
shown that there is a statistic difference in the case of AC group (132,3 ± 9,5 mg/100 mg.
tissue), with a decrease of 21,3 %, (p < 0,01), comparative to C group (168,1 ±12 mg/100 mg.
tissue).
The MB group had a moderate decrease, statistically insignificant, of the average protein
quantity (157,8 ± 5,9; - 6,2 %), comparable to C group. Average values of the total protein
quantity of fetal brain omogenate, percentage differences between groups, and the statistic
significance of the results, are presented in table 3.
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Table 3
Average values of total protein quantity in fetal brain homogenate (mg/g tissue)
Group
n
X ± SE
D%
p
C
8
168,1 ±12
-
AC
8
132,3 ± 9,5
- 21,3
< 0,01
MB
8
157,8 ± 9,4
- 6,2
> 0,3
DISCUSIONS
The first objective of our works was to study the embryotoxic actions of MSG, ASP and
CYS upon some morphological and functional gestational parametters in rabbit fetuses, and
the synergic and the presumable protective effects of some antioxidant micronutrients against
MSG, ASP and CYS treatment during organogenetic period of gestation in rats.
We have considered of importance the study of this particular moment of this
onthogenetic development, because the data gathered from other studies on the action that
these amino acid has on mammals’ organism during the phase of intrauterine development are
very sporadic.
At the same time we tried to attenuate or even counteract the noxious, embryotoxic effects
of these amino acid, with the help of some compounds with a protective, antioxidant
potential: the vitamin-mineral organic complex.
The study of data gathered from literature showed us that the problems of the action that
excitotoxic amino acids have on mammalian organism, respectively on humans, is still an
important issue, and finding solutions for attenuating their nocuous effects is one of the
preoccupations of many agencies for health protection, as well as laboratories from all over
the world.
In this context, our research is within the area of investigations done in countries with a
tradition in this field, and our study’s approach concerning the protection of embryonic
development under conditions of maternal consumption of some excitotoxins, namely Lmonosodium glutamate L-aspartate and L-cysteine (amino acids that enter in the composition
of hydrolyzed proteins (FDA, 2010), has an original character, totally new, unreported in
literature.
The issue is of great interest, from the point of view of human health, considering that
pregnant women consume a highly varied array of aliments that contain MSG, ASP and CYS
too, used as vegetable hydrolysed proteins food additives.
Glutamate and aspartate are a powerful amino acid neurotransmitters that clearly plays a
pivotal role in neuronal differentiation, migration and survival in the developing brain.
Glutamate is also very important for the neuronal plasticity, in the formation of synapses and
neuronal circuitry, long-term potentiation and depression, and both normal learning and
addictive behavior, but in experimental condition of elevated level in the brain, such as after
systemic administration, MSG generally produced brain lesions in immature animals without
a fully developped blood-brain barrier, (Goldsmith, 2000; Gao et al., 1994; Meldrum, 2000).
The studies of Fernstrom, (2001), and Lau and Tymiansky, (2010). revealed that after the
treatment with MSG in the gestational period, the infant rodents are born with incomplete
myelininization. This amino acid affects all the developping brain, the hypothalamic regions
that lack blood brain barriers, the extrahypothalamic regions (hippocampus, choroid plexus
and cortex), because the developping brain are hypervulnerable to the excitotoxic insults.
116
These aspects cause very important consequences on the following prepubertal, juvenile
stages of developement of animals.
Excitotoxins are molecules, such as MSG and aspartate, that act as excitatory
neurotransmitters, and can lead to neurotoxicity when used in excess. These substances,
usually amino acids, that react with specialized receptors in the brain in such a way as to lead
to destruction of certain types of brain cells. Glutamate is one of the more commonly known
excitotoxins. MSG, the sodium salt of the amino acid glutamic acid or glutamate, is an
additive used to enhance the flavor of certain foods. It does not have a flavor of its own, but is
believed to enhance the taste of other foods by stimulating glutamate receptors on the tongue,
(FDA, 2010; Lee et al., 2010; Samuels, 2010).
With the discovery of excitatory amino acid (EAA) transmitter systems and identification
of EAA receptor subtypes (N-methyl-d-aspartame [NMDA], kainic acid, and amino-3hydroxy-5-methyl-isoxazole-4-proprionic acid) and their antagonists, it has become widely
accepted that glutamate, aspartate, and other environmental substances have neurotoxic
(excitotoxic) effects in the human nervous system, (Henneberry et al.,1989; Olney, 1994).
The adverse reactions to MSG have been theorized to be due to MSG's actions at
glutamate receptors in glutamate-responsive tissues.
The glutamate/aspartate is unlikely to cross the blood-brain barrier, and very few data are
available showing that the amounts of these amino acids have significant neural effects.
Glutamate and aspartate crosses the blood-brain and placental barrier only by active transport,
and concentrations in the brain are kept low and independent of plasma concentrations.
However, glutamate and aspartate freely enters brain regions that lack blood-brain barriers
(circumventricular organs, e.g., the hypothalamus). It has been shown that glutamate can
destroy circumventricular organ neurons by an excitotoxic mechanism (via the NMDA
receptor) in all animal models appropriately tested (cats, chickens, guinea pigs, hamsters,
mice, monkeys, rabbits). In fact, much of the research performed proving that glutamate was
safe for human consumption may have been flawed. Tests using infant monkeys anesthetized
these animals with phencyclidine, now known to inhibit the neurotoxic effects of glutamate
on the hypothalamic neurons by its potent antagonism of the specific subtype of NMDA
receptor, (Puica et al., 2004, 2007a,b, 2008; Smith et al., 2003).
Embryonic development is characterized by three main interrelated processes:
differentiation, morphogenesis and growth. There are several fundamental mechanisms that
guide these processes and in sure synchronized and orderly development of the embryo. In the
present, it is been established that morphogenetic cell movements in the embryo occur on
performed matrices, e.g., early collagen, onto which the cells are attached during their
migration. The developing embryo and fetus are more sensitive during the whole prenatal
period in utero. Cell proliferation, differentiation and migration are the determining processes
during the prenatal development from the fertilized oocyte (zygote) to the fetus at birth.
Foreign substances can interfere with all three events but the embryo-sensitivity varies during
the developmental stages and with respect to various biological effects. A derangement of
these extensive and complex cellular movements may lead to abnormalities in morphogenesis.
The normal growth of the embryo or an organ involves an increase in either the number of
cells (total DNA) or the size of the cell (protein/DNA ratio), or both, (Blaylock, 1997;
Iconomidou et al., 1999).
The results of the histochemical study revealed that administration of MSG, ASP and
CYS during organogenetic period of gestation caused for rabbit fetuses, a diminution of green
fluorescence exhibited by native DNA, respectively a diminution of the number of nuclei, as
well as that of the red intracellular fluorescence emitted by the native RNA under UV light, in
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all of the cerebral structures, as well as the apparition of some more extended pericellar,
extracellular ‘beaches’ that had red fluorescence (denatured DNA), in the case of groups
treated with the higher glutamate dose.
The histochemical study, done after staining with AO of brain sections and microscopic
examination under UV, reveals the green fluorescence exhibited by the native, nuclear DNA,
the red fluorescence emitted by the native cytoplasmatic and nucleolar RNA, and the red
fluorescence from the pericellular areas, that is characteristic for denatured nuclear DNA. The
green fluorescence represents the condensation of the nuclear heterochromatin, and the red
one the condensation of the cytoplasmatic content with AO, resulting metachromatic
precipitates that are visible under UV, so the sensitivity of DNA for the formation of
precipitates is modulated by the interaction of the staining agent with the nuclear proteins, and
that of the RNA is modulated by the interaction of the AO with the cytoplasmatic formations,
this reaction revealing DNA and RNA of viable cells (Darzynkyewicz, Traganos, and
Sharpless, 1975; Evenson et al, 1991).
Using fluorescent staining, we showed that MSG, ASP and CYS exposure can cause DNA
strand breaks, chromatin condensation, nuclear fragmentation, and DNA laddering. The loss
of green fluorescence for DNA and the red one for RNA, respectively cellular death (caused
both by apoptosis and the action of some toxic substances), corresponds with a condensation
followed by lysis of the nuclear chromatin (phenomena of necrosis and cellular apoptosis),
(Guettiter and Ziol, 1998).
The same authors observed that the red extracytoplasmatic fluorescence (pericellular), is
determined by the denaturizing of the DNA, respectively by cellular necrosis.
Sensibility to precipitation of DNA and RNA in situ is modulated by the interaction of
AO with nuclear proteins, respectively with nucleolar and cytoplasmatic proteins, this
reaction being proof of nuclear chromatin, and finally of cellular viability. Thus,
histochemical staining of unfixed sections with Acridin orange G also represents a test of
cellular viability (Evenson et al, 1991).
The prenatal administration of glutamate, aspartate and cysteine also induces the alteration
of the endothelial structures of the choroidian plexes, with a diminution of the green
fluorescence exhibited by the native nuclear DNA of the endothelial and cells and that of the
pericytes, associated with a generalised disorganisation of the cellular chords, as well as with
the presence of red extracellular fluorescence beaches, with a possible effect the
morphofunctional alteration of the hematoencephalic barriers and of the cerebrospinal fluid.
Our results revealed that during the prenatal stage the cerebral structures: the cortical,
hippocampic, hypothalamic and those of the choroidian plexes are extremely vulnerable to the
excitotoxic action of the glutamate.
AO staining being proof of cellular viability, the diminution of green fluorescence emitted
by DNA in our experiments on rabbits, actually represents a diminution of the number of
viable cells both in the cerebral cortex, the hippocampus (areas CA1, CA2, CA3 and the
dentate gyrus) and the periventricular hypothalamic areas, especially in the nucleus arcuatus
of hyppothalamus (ARC).
The histochemical examinations revealed numerous apoptotic cells characterized by
nuclear fragmentation and condensation were observed in the hippocampus of MSG, ASP and
CYS-treated fetuses with the highest number seen in the CA1 and CA2 areas. There were
seen significantly decreased density of hippocampal pyramidal cells in the CA1 and CA3
areas, especialy. Our results are in agreement to literature data, (Cristescu et al., 2004,
Cristescu, 2007).
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There was been established that mammalian brain shows pronounced sensitivity to longterm damage due to prolonged cell formation and maturation processes. This entails extended
chains of responses going along with the natural course of development. Hierarchical levels of
successive responses are represented by (i) initial effects in the substrate present at the time of
exposure, (ii) secondary effects in daughter cells and structures being formed immediately
after exposure, and (iii) long term responses of the later cell progeny, or of premature cerebral
subunits, respectively. The response-chains can interfere with a variety of compensatory
responses. End-points of effects are unlikely to be reached before the brain achieves structural
and functional maturity, (Abe et al, 1992; Cristescu et al., 2004; Greenamyre and Higgins,
1997; Kobe, 2001; Puica, 1997; Van den Pol et al, 1994).
It is well known that the development of the brain is a very complex process, that takes
place in space-temporal ambiance, that is under close control by biochemical, structural and
neurophysiological events. The most subtle alterations of these parameter can lead to
functional alterations that cause further structural and behavioral alterations. The etiology of
excitotoxin induced developmental damage is characterized by response-chains with
hierarchical levels of neurological defects, which are particularly expressed in the mammalian
brain during its prolonged and complicated genesis. After non-lethal exposure to excitotoxins,
response-chains go along with the natural course of development. This implies that
manifestation of damage shifts to increasingly higher levels of biological organization before
end-points are reached during postnatal life. Numerous studies of developmental excitotoxin
effects, specifically on the brain of rats and mice, permit a systematic study of the transfer of
effects and of concurrent compensation responses. Thus, initial effects to directly exposed
cells, secondary effects to daughter cells and related tissues, long-term effects to the later cell
progeny and maturing tissues, as well as end-points in the mature brain can be differentiated,
(Blaylock, 2002).
Neurodegeneration can be defined as the abnormal loss of neurons in a developing or
mature animal. While neurodegeneration has been recognized as the cause of abnormal
central nervous system (CNS) function in humans and experimental animals for many
decades, the wide variety of physiological insults resulting in death of differentiated neurons
obscured possible similarities in effector mechanisms among these myriad disorders,
(Blaylock, 1997).
It has been proposed that exposure to excitotoxins in humans (such as glutamate, aspartate
and cysteine) during fetal life may cause alterations in brain development that could later
result in such serious brain disorders as autism, learning disorders, hyperactive behavior, and
possibly schizophrenia. In general, the baby's brain is most vulnerable to gross malformations
during the first period of embryonic development. This would mean that the areas with a more
immature barrier would be infinitely more susceptible to the effects of excitotoxins in the
food since they could easily enter that portion of the brain. In the case of the hypothalamus,
this could result in severe derangements of the endocrine control mechanisms. Indeed this is
what has been seen in a multitude of animal studies, (Blaylock, 1997, 2002; Ikeda, 2003;
Massie et al., 2003).
The neurons of the circumventricular hypothalamic area and those of the hippocampus
from the immature brain are also hypervulnerable to the destructive action of excitotoxins,
respectively of glutamate, aspartate and cysteine. The inner reason of this vulnerability is not
yet sufficiently clear, the only known thing is that the receptor subtypes for the immature
CNS that mediate the neuronal destructions are hypersensitive to the excitotoxic stimulation.
The concern of excitotoxins (e.g. glutamate) ingestion during pregnancy is not limited to
obvious birth defects, but changes or damage to certain areas of the brain. The potential
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damage includes parts of the brain involved in complex learning as well as hormonal control
(e.g., hypothalamus), (Barb et el, 1996; Blaylock, 1997; Stengik et al, 1982).
The experimental data of Goldsmith (2000) has shown that the high plasmatic level of
glutamate for gestating mice and for new-born mice, caused by the subcutaneous injection
single doses of 0.1-0.5 mg/g body weight of MSG, can cause selective neuronal destruction in
the new-born mice’s brain, the periventricular hypothalamic area and especially the
ventromedial area of the arcuate nucleus, as well as the structures of the median eminence,
incompletely matured in this development stage are extremely sensitive to the neurotoxic
action of this amino acid. Administration of larger doses (0.3 – 0.5 mg/g body weight) of
glutamate caused lesions of a higher number of neurons from the above mentioned areas, but
also from the neighbour areas of the hypothalamus, clearly indicating that glutamate
penetrates all structures of the developing brain.
Dr.Olney had shown in 1972 that large doses of glutamate given to pregnant rhesus
monkeys late in gestation also resulted in damage to the fetus brain. Adults, including
pregnant women, ftequently ingest large concentrations of MSG and other excitotoxins in
their food. In one study it was found that some restaurants add as much as 9.9 grams of MSG
to a single dish, enough to produce brain damage in experimental animals. Liquids containing
MSG, such as soups, are absorbed faster and more completely than solid foods. Experiments
have shown that MSG in a liquid form is much more toxic to the brain than when included in
solid food. Also, humans have been found to have higher blood levels of glutamate following
ingestion of MSG than does any other animal species.
Several experiments have demonstrated that under such conditions, glutamate can by-pass
the barrier systems and enter the brain in toxic concentrations. Gao (1994) demonstrated that
the administration of MSG in several stages of mice gestation, causes structural and
functional alterations in several specific areas of the brain (cortex, hypothalamus) in the
immediately postnatal phases of development, thus proving a transplacental, neurotoxic effect
of this amino acid.
Olney (1994) has proven that L-cistein (CYS) – homologue of MSG and L-aspartate of
sodium, but with a higher neuroexcytotoxic effect, has the same contribution as other
endogenous excytotoxins in causing neurodegenerative aspects in the early stages of
ontogenetic developments of lab animals.
All these fluorescent histochemical changes registered in our experiment were prevented
by the antioxidant micronutrients administration during organogenetic period of gestation. In
this way, treatment of gestating rabbits with GLU, ASP and CYS associated with the
medicinal bioactive compounds magnesium in contrast with the excitotoxic action of MSG,
ASP and CYS on the developing organism, had as a prime result the reduction of the number
of cells with lesions, in the fetal brain of rabbits, results that were confirmed by the
histochemical studies.
Biochemical investigations. Treatment during the organogenetic stage of gestation of
animals with GLU, ASP and CYS, caused a significant diminution of DNA and total
proteins quantities from fetal brain homogenate. There is also a perfect consistency between
the alterations of body weight, of tissue and internal organs weight, the morphopathological
aspects described in the histological study of the brain structures (data non presented), as well
as with the qualitative and quantitative determinations of nucleic acids and proteins. The
significant diminution of the relative number of nuclei, respectively of neuronal, viable cells,
from the structures of the CNS that we analyzed, correlated with the quantitative decrease of
DNA, as well as that of proteins obtained from brain homogenate, show once more the
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inhibitory effect of MSG, ASP and CYS over the synthesis of nucleic acids, respectively over
protein biosynthesis.
As we have mentioned above, there are relatively few data in literature that describe the
negative effects of these excitotoxic amino acids treatment over embryonic and fetal
development.
A high level of protein synthesis occurs during embryonic development: growth and
differentiation depend on the availability of specific proteins. The protein deprivation and the
inhibition of protein synthesis after by MSG, ASP and CYS treatment induced a significant
increase in the incidence of fetal resorbtions in rats (data non presented). Studies in the chick
and in nthe rat (Persaud, 1974) have shown that embryonic development is severely impaired
following treatment with excitatory amino-acid analogues.
According to some authors, excitotoxic amino acids, so glutamate/aspartate as well, could
even block, in certain situations, the biosynthesis of nucleic acids and peptides (Ankacrona et
al., 1995; Finiels et al. 2002).
Kure et al (1991) have shown from in vitro cultures of astrocites from the hippocampus
that MSG can induce the internucleosomal cleavage of DNA, but the DNA fragmentation and
neuronal death can be prevented by inhibiting the activity of the endonuclease.
Research of Reynolds et al, (1995) has proven that MSG plays an important part in the
developing brain, by exerting a regulatory influence on the genesis of synapses and neuronal
development, thus sustaining the growth, differentiation and maturation of cells in the CNS,
as well as the general growth of the organism. The superactivation of neurons through
excessive levels of MSG is responsible for excitotoxic alterations at a subcellular level
(nucleus, cellular organelles), of the developing neurons, with serious repercussions on the
future development of organisms.
The administration, in our experiment, of GLU, ASP and CYS associed with the organic
antioxidant complex caused the lessening of the harmful effects of these excitotoxic amino
acids administration over DNA and protein biosynthesis. The administration of these
bioactive compounds caused an immediate, more efficient protection of the hypothalamic and
hypophysial nervous cells, respectively of the biosynthesis of nucleic acids and proteins,
against the excitoneurotoxic effect of MSG, ASP and CYS.
CONCLUSIONS
• The treatment with L-monosodium glutamate, L-aspartate and L-cysteine during 6-18
days of gestation in pregnant rabbits caused morphostructural alteration of all the fetal
cerebral regions, revealed by decrease of the DNA and RNA fluorescence of the neurons, and
by the inhibition of the proteic biosynthesis of nucleic acids in the brain homogenate.
• These data demonstrate the toxic, transplacental effect of these amino acids on the
organism, during this stage of fetal development.
• The simultaneous administration of MSG, ASP, CYS associd with a bioactive
antioxidant complex ad in entire period of gestation in pregnant rabbits, caused a protective
effect for fetuses, over all the histochemical and biochemical parameters that were analyzed.
• Our data demonstrate the extreme vulnerability of organisms, during the intrauterine
development, towards the toxic effects induced by the administration of MSG, ASP and CYS,
during organogenetic stage of gestation. The organic complex proved to be efficacious for
attenuating the excitotoxic effects of MSG, ASP and CYS.
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